Multi-omics analysis identifies COX6A2/SCF/c-Kit-mediated mitochondrial remodeling as a key mechanism of Tangwei’an decoction in diabetic gastroparesis

Abstract

BACKGROUND

Tangwei’an decoction (TWAD) is a traditional Chinese medicinal formula derived from classical prescriptions including Zhizhu Decoction, Xiaobanxia Decoction, and Dahuang Huanglian Xiexin Decoction, and is clinically used for the treatment of diabetic gastroparesis (DGP). However, its pharmacological basis and underlying mechanisms remain unclear.

AIM

To systematically elucidate the bioactive constituents and therapeutic mechanisms of TWAD in DGP using an integrated strategy combining serum pharmacochemistry, integrated proteomics and metabolomics, and in vivo and in vitro experimental validation.

METHODS

Ultra-performance liquid chromatography hybrid quadrupole-Orbitrap high-resolution mass spectrometry was used to investigate the chemical components of TWAD. As a DGP model, db/db mice were given either water or TWAD every day for eight weeks. Integrated proteomics and metabolomics were used to investigate the mechanisms behind TWAD-mediated improvement of gastrointestinal function. Mouse gastric smooth muscle cells were used for complementary in vitro mechanistic validation, and key pathway nodes were verified in both gastric tissue and smooth muscle cell assays.

RESULTS

Ultra-performance liquid chromatography coupled with a hybrid quadrupole-Orbitrap high-resolution mass spectrometer identified 93 constituents in TWAD, and 32 compounds were detected in TWAD-containing serum (9 prototypes and 23 metabolites). In db/db mice with DGP phenotypes, TWAD improved gastric emptying and gastrointestinal motility and attenuated gastric pathological injury, without significantly affecting body weight or fasting glucose. Integrated proteomics and metabolomics consistently prioritized oxidative phosphorylation as the dominant TWAD-responsive pathway, with COX6A2 emerging as a key regulated node. In vivo and gastric smooth muscle cell assays further supported that TWAD modulates mitochondrial dynamics via the COX6A2/SCF/c-Kit axis.

CONCLUSION

TWAD alleviates DGP by restoring mitochondrial dynamics through the COX6A2/SCF/c-Kit signaling pathway. This effect is associated with improved oxidative phosphorylation and gastrointestinal motility in vivo and in vitro, supporting TWAD as a potential therapeutic strategy for DGP.

Key Words: Tangwei’an decoction; Diabetic gastroparesis; Multi-omics; COX6A2/SCF/c-Kit; Mitochondrial dynamics

Core Tip: Tangwei’an decoction (TWAD) is clinically used for diabetic gastroparesis (DGP), but its mechanism remains unclear. This study combined serum pharmacochemistry, integrated proteomics and metabolomics, and experimental validation to show that TWAD alleviates DGP by restoring mitochondrial dynamics. Oxidative phosphorylation was identified as the major pathway regulated by TWAD, with COX6A2 as a key target. TWAD also modulated the SCF/c-Kit axis, providing mechanistic support for its therapeutic potential in DGP.

INTRODUCTION

Diabetes has expanded dramatically over the past three decades, with an estimated 828 million adults aged 18 years and older living with diabetes worldwide in 2022, an increase of 630 million since 1990[1]. Diabetic gastroparesis (DGP) is a clinically significant diabetes complication characterized by delayed gastric emptying (GE) in the absence of mechanical obstruction, early satiety, postprandial fullness, nausea and vomiting, decreased oral intake, and an increased risk of malnutrition[2,3]. Chronic hyperglycemia and glycemic fluctuations can disrupt gastric motility through neuropathic injury and oxidative stress, thereby contributing to delayed GE[4]. A recent systematic review and meta-analysis estimated a pooled prevalence of 9.3% for gastroparesis among adults with diabetes, with significant variations by diabetes type and study methodology[5]. Beyond the burden of symptoms, DGP modifies the timing of nutrient delivery as well as the pharmacokinetics and absorption of oral medications, which increases glycemic variability and symptom-driven healthcare consumption[6,7]. To relieve gastrointestinal symptoms, improve glycaemic control and quality of life in patients with DGP, and reduce the growing societal and economic burden, effective and comprehensive prevention and management strategies are urgently needed.

The pathophysiology of DGP is multifactorial and remains incompletely defined. Chronic hyperglycemia and glycemic variability can disrupt gastric motor function via neurohumoral dysregulation, oxidative stress, and smooth muscle dysfunction, resulting in delayed GE[8]. Recent studies further indicates that DGP progression is associated with injury to interstitial cells of Cajal (ICCs) and gastric smooth muscle cells (GSMCs), partly driven by gut microbiota dysbiosis and impaired cellular homeostasis[9]. Mitochondrial bioenergetic impairment has been linked to delayed GE in diabetic gastroenteropathy and is characterized by dysregulated oxidative phosphorylation (OXPHOS) programs and reduced mitochondrial capacity[10,11]. In parallel, SCF/c-Kit activity mediates gastrointestinal motility signal transduction, and its dysregulation contributes to peristaltic dysfunction[12]. However, the mitochondrial determinants that connect OXPHOS disturbance to impaired motility signaling in DGP remain to be elucidated.

Current therapeutic options for DGP remain limited and still rely largely on prokinetic agents alongside glycaemic management, with frequent challenges such as incomplete symptom relief and relapse after treatment discontinuation[13]. Traditional Chinese medicine (TCM) formulae may be well suited to DGP as inherently multi-component interventions that engage multiple targets[14-16]. TCM may improve DGP through coordinated protection of ICCs and GSMCs, regulation of brain-gut peptides and neuropathy, attenuation of oxidative stress and inflammatory injury, restoration of gut microbiota homeostasis, and modulation of ICC autophagy-apoptosis crosstalk[9,17]. Tangwei’an decoction (TWAD) was developed by Professor Xiao-Lin Tong based on more than three decades of clinical experience in diabetes and its complications, guided by the principle of strengthening the spleen, regulating qi, and harmonizing the stomach, and formulated through modification of classical prescriptions including Zhizhu Decoction, Xiaobanxia Decoction, and Dahuang Huanglian Xiexin Decoction. Previous research in diabetic rats found that TWAD-based interventions improved gastrointestinal dysmotility in a dose-dependent manner, as well as reducing AGE/RAGE signaling, restoring SCF and c-Kit, normalizing neuroendocrine hormones, and partially reversing morphometric and biomechanical remodeling of the gastrointestinal wall[18,19]. Nevertheless, the molecular basis for TWAD’s prokinetic effects and its upstream metabolic drivers remain insufficiently defined. To elucidate the mechanisms by which TWAD ameliorates DGP, we combined chemical profiling using ultra-performance liquid chromatography coupled with a hybrid quadrupole-Orbitrap high-resolution mass spectrometer (UHPLC-Q-Orbitrap HRMS) with integrated proteomics and untargeted metabolomics of mouse gastric tissues, followed by in vivo and in vitro mechanistic validation to identify key targets and pathways, thus establishing a scientific basis for its clinical application.

MATERIALS AND METHODS

Preparation and analysis of TWAD

TWAD consists of eight TCMs (Table 1), all purchased from Guang’anmen Hospital, Chinese Academy of TCM, and authenticated by a licensed pharmacist. The herbs were soaked in cold water for 30 minutes (2-3 cm above the surface) and decocted in an earthenware pot. The mixture was first boiled and then simmered for 30 minutes with the lid closed and occasional stirring. The extract was filtered, and the residues were decocted again using the same procedure. Each extraction yielded approximately 200 mL of solution. The two extracts were combined and centrifuged at 3500 rpm for 10 minutes. The supernatant was concentrated to 3 g/mL using a rotary evaporator and subsequently freeze-dried with a vacuum-assisted device (Biocool, China) to obtain a lyophilized powder. The SD rats (n = 3) were given orally a single dose of TWAD (11.87 g/kg) and blood samples were collected at 0 hour, 1 hour, 2 hours and 4 hours. The serum collected at these time points was then mixed and used for subsequent intervention experiments.

Table 1 The composition of Tangwei’an decoction.

Chinese name	Latin name	Medicinal parts	Dosage/g
Zhishi	Citrus aurantium L.	Immature fruit	15
Baizhu	Atractylodes macrocephala Koidz.	Rhizome	15
Binlang	Areca catechu L.	Seed	6
Dahuang	Rheum officinale Baill.	Root and rhizome	6
Banxia	Pinellia ternata (Thunb.) Breit.	Tuber	9
Shengjiang	Zingiber officinale Rosc.	Rhizome	10
Taoren	Prunus persica (L.) Batsch	Seed	6
Dangshen	Codonopsis pilosula (Franch.) Nannf.	Root	15

The chemical components of TWAD were analyzed by UHPLC-Q-Orbitrap HRMS as follows. TWAD extract was prepared by dissolving 1.0 g lyophilized powder in 40 mL 80% methanol, ultrasonicated for 30 minutes, and centrifuged (12000 rpm, 4 °C, 10 minutes); 100 μL of the supernatant was used for injection. Serum samples (100 μL) were mixed with 300 μL methanol, vortexed, and centrifuged under the same conditions. The supernatant was dried by vacuum centrifugation, reconstituted in 90 μL 50% methanol, vortexed, centrifuged again, and 80 μL was transferred for analysis.

UHPLC separation was performed on a Vanquish Flex system (Thermo Fisher Scientific, United States) with an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.7 μm; Waters, United States). The mobile phase was water (0.1% formic acid, A) and acetonitrile (B) at 0.3 mL/minute, column temperature 40 °C, and injection volume 6 μL.

Mass spectrometry was carried out on a Q Exactive orbitrap MS (Thermo Fisher Scientific, United States) with HESI-II source. Parameters were: Spray voltage 3.7/3.5 kV (positive/negative), capillary 320 °C, sheath/auxiliary gas 30/10 psi, desolvation 300 °C, nitrogen as collision gas (1.5 mTorr). Data were acquired in full scan/dd-MS² mode (full scan: Resolution 70000, AGC 1 × 10⁶; dd-MS²: Resolution 17500, AGC 1 × 10⁵, top 10, isolation window m/z 2, stepped collision energy 10/30/60 eV). Data were processed using Progenesis QI 3.0, and compounds were identified by retention time, mass accuracy, fragment matching, isotope distribution, and database searches (TCM Pro 2.0 and public databases).

Experimental animals

A total of 55 male C57 BLKS/J db/db mice with spontaneous diabetes and 13 male C57 BLKS/J db/m mice (8 weeks old) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (license number: SCXK [Su] 2023-0009). All animals were housed in the Laboratory Animal Center of Beijing Pharmacokang Biotechnology Co., Ltd. under specific pathogen-free conditions, with controlled temperature (23 ± 2 °C), relative humidity (50%-60%), and a 12-hour light/dark cycle (lights on from 08: 00 to 20: 00). All procedures were conducted in strict accordance with the Regulations on the Administration of Laboratory Animals issued by the National Science and Technology Commission of China and were approved by the Institutional Animal Care and Use Committee of Beijing Pharmacokang Biotechnology Co., Ltd., No. GPT-BJAP20240328-1.

Establishment and treatment of DGP mouse model

After one week of acclimatization, body weight, fasting blood glucose, GE, and small intestinal transit were assessed in male C57BLKS/J db/db mice and age-matched db/m littermates maintained under standard conditions. Fasting blood glucose was measured using a glucometer after 6 hours of fasting. Db/db mice showing markedly elevated fasting blood glucose together with significantly reduced GE and small intestinal transit relative to db/m controls were considered to have successfully developed DGP[20].

Following model establishment, 50 male db/db mice were randomly assigned into 5 groups (n = 10 per group) using a random number table: Model group, TWAD high-dose group (TWA-H), TWAD medium-dose group (TWA-M), TWAD low-dose group (TWA-L), and Mosapride group, while 8 db/m mice served as the Control group. The clinical daily dose of TWAD was 82 g/day (crude drug) for a 70-kg adult. Based on a human-to-mouse body surface area conversion ratio of 1:9.1, the equivalent mouse dose was calculated to be 10.66 g/kg/day and used as the medium dose, while the high and low doses were set at 21.32 g/kg/day and 5.33 g/kg/day. Mosapride citrate was administered at 1.95 mg/kg/day. All groups received daily oral gavage for 8 consecutive weeks: Control and Model groups were administered distilled water (0.1 mL/10 g body weight), TWA-H, TWA-M, and TWA-L groups received TWA at 21.32, 10.66, and 5.33 g/kg/day, respectively, and the Mosapride group received Mosapride citrate at 1.95 mg/kg/day.

Measurement of GE rate and intestinal transit

After the final gavage, all mice were fasted for 16 hours with free access to water and orally administered 0.1 mL/10 g body weight of 1.5% carboxymethylcellulose containing 0.05% phenol red. Blood was drawn from the orbital venous plexus 30 minutes after gavage, and the mice were euthanized by cervical dislocation. The abdominal cavity was subsequently opened. The stomach was ligated at the cardia and pylorus and excised for weighing after blotting dry as the total stomach weight. The stomach was then opened along the greater curvature to expose the gastric contents, rinsed with phosphate buffered saline (PBS), blotted dry, and weighed again as the net stomach weight. GE was then calculated based on the following formula: GE (%) = [1 – (total stomach weight - net stomach weight)/gavage volume] × 100%[21].

The small intestine was excised, straightened, and laid on white filter paper, and the distance traveled by phenol red from the pylorus (L2) and the total length of the small intestine (L1) were measured to calculate intestinal transit: Intestinal transit (%) = (L2/L1) × 100%. The furthest reach of phenol red was confirmed by making a small incision at the distal end and adding drops of 0.5 mmol/L NaOH, with a color change indicating the presence of phenol red.

Histopathological examination

Paraffin-embedded tissue sections were deparaffinized in xylene, hydrated through graded ethanol, stained with hematoxylin and eosin, dehydrated, cleared in xylene, and mounted. Sections were observed and imaged under an optical microscope. Gastric histopathology was scored as follows. Inflammatory infiltration was scored as follows: 0, few inflammatory cells confined to the superficial mucosa; 1, denser inflammatory cells involving more than one-third of the mucosal layer; 2, dense inflammatory cells involving the entire mucosal layer; 3, marked neutrophil infiltration with visible small abscess-like lesions. Mucosal/glandular damage was scored as follows: 0, normal mucosa; 1, epithelial cell damage; 2, glandular cell damage; 3, mucosal erosion, hemorrhage, or ulceration[22]. The total gastric histopathological score ranged from 0 to 6.

Colonic histopathology was scored as follows. Inflammatory infiltration was scored as follows: 0, minimal inflammatory cell infiltration; 1, mild inflammatory cell infiltration; 2, moderate inflammatory cell infiltration; 3, severe inflammatory cell infiltration. Tissue damage was scored as follows: 0, intact mucosa; 1, mild epithelial injury; 2, moderate crypt damage and goblet cell loss; 3, severe mucosal destruction with marked crypt disruption. Architectural disruption of the mucosa was scored as follows: 0, normal architecture; 1, mild distortion; 2, moderate disruption; 3, severe disruption[23]. The total colonic histopathological score ranged from 0 to 9.

Serum gastrointestinal hormone assays

Blood samples were allowed to clot at room temperature for 1 hour and centrifuged at 3500 r/minute for 15 minute at 4 °C. Serum was stored at -80 °C. Levels of motilin, cholecystokinin (CCK, Jianglai Bio, JL10451, lot: 202312, Shanghai, China), serotonin (5-HT, Elabscience, E-EL-0033, lot: UX0966N22855, Wuhan, China), vasoactive intestinal peptide (VIP, Elabscience, E-EL-M1234, lot: UX10R4R11106, Wuhan, China), and substance P (SP, Elabscience, E-EL-0067, lot: UX1315124156, Wuhan, China) were measured using ELISA according to the manufacturer’s instructions. Absorbance was read at 450 nm.

Proteomics analysis of gastric tissue

Total protein was extracted from frozen gastric tissues (approximately 100 mg; n = 5 per group: Control, Model, TWA 21.32 g/kg) using a phenol-based method. Tissues were pulverized in liquid nitrogen, homogenized in phenol buffer, and proteins were precipitated, washed, and solubilized. After quantification, 50 μg protein per sample was reduced, alkylated, and digested with trypsin (1:50) overnight. Peptides were desalted and reconstituted with iRT markers for DIA proteomic analysis.

Proteomic analysis was conducted by Shanghai OE Biotech Co., Ltd. using a nanoElute2 LC system (Bruker, United States) coupled to a TimsTOF Pro2 mass spectrometer with a CaptiveSpray ion source. Approximately 300 ng of peptides were separated on a 15-cm C18 column (1.9 μm, 100 μm i.d.) with a 10-minute gradient of 6%-80% acetonitrile (0.1% formic acid) at 400-700 nL/minute. The diaPASEF method was implemented with the following parameters: Capillary voltage, 1.6 kV; dry gas, 3.2 L/minute at 180 °C; MS range, 300-1500 m/z; and collision energy ramped from 59 eV (1/K0 = 1.3 Vs/cm²) to 20 eV (1/K0 = 0.7 Vs/cm²). Instrument control and data acquisition were performed using timsControl software (v6, Bruker, United States). The DIA raw data were processed using Spectronaut Pulsar 18.4 (Biognosys, Switzerland) with the UniProt-Mus musculus-10090-2024.2.1.fasta database for peptide and protein identification. Differentially expressed proteins (DEPs) with a P value < 0.05 and an FC ≥ 2.0 were eliminated for additional bioinformatics research using statistical analysis after the quantitative findings were standardized. Additionally, DEPs were employed for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis.

Metabolomics analysis of gastric tissue

Gastric tissue samples (25 mg) were homogenized in 500 μL of extraction solution containing internal standard (methanol:acetonitrile:water = 2:2:1, v/v/v) using a 4-minute grinding treatment at 35 Hz followed by 5-minute ice-water bath sonication. After three repeated cycles of homogenization, samples were stored at -40 °C for 1 hour, then centrifuged at 12000 rpm for 15 minutes at 4 °C. The supernatant was transferred to new vials for liquid chromatography-tandem mass spectrometry analysis, with quality control samples prepared by pooling equal volumes of supernatant from all gastric tissue extracts.

Liquid chromatography-tandem mass spectrometry analysis was performed on a UHPLC system (Waters ACQUITY UPLC I-Class Plus, United States). Raw data were converted to mzXML format using ProteoWizard and processed through an in-house R package based on XCMS for peak detection, alignment, and integration. Metabolite annotation was conducted against a custom spectral database with a match score cutoff set at 0.3. Data processing was further performed using Progenesis QI V2.3, including baseline filtering, peak identification, retention time correction, and normalization. Identification criteria included 5 ppm precursor mass error, 10 ppm fragment mass error, and 5% fragment ion threshold, with reference to HMDB, LipidMaps (v2.3), METLIN, and a self-built database. Peaks with > 50% missing values were excluded, and zero values were replaced with half of the minimum value. Compounds with identification scores below 36 were removed, and data from positive and negative ionization modes were merged into a unified matrix.

Multivariate statistical analysis included principal component analysis (PCA) to evaluate sample distribution and analytical stability, along with OPLS-DA and PLS-DA to identify group-discriminant metabolites. Model quality was assessed using 7-fold cross-validation and 200 permutation tests. Metabolites with VIP > 1.0 from the OPLS-DA model and P < 0.05 from Student’s t-test were considered significantly altered. These differential metabolites were subsequently subjected to KEGG pathway enrichment analysis.

Correlation analysis of proteomics and metabolomics

To investigate the interrelationships between proteomic and metabolomic alterations in DGP, we conducted a correlation analysis integrating differentially expressed metabolites (DEMs) and DEPs. Significant associations were screened using thresholds of |correlation coefficient| > 0.8 and P < 0.05. The interaction patterns between DEMs and DEPs were visualized using a correlation heatmap. Furthermore, co-enriched KEGG pathways were constructed based on the significantly correlated DEMs-DEPs pairs to identify key regulatory networks involved in DGP pathophysiology.

Cell culture and treatment

Primary mouse GSMCs (MGSMCs) were obtained from Procell Biotechnology Co., Ltd. (CP-M237) and maintained in MGSMC complete medium (CM-M237) at 37 °C in a humidified incubator with 5% CO₂. According to the manufacturer’s product specification, these cells were identified by α-SMA immunofluorescence staining, with a purity of more than 90%, and were free of human immunodeficiency virus type 1, hepatitis B virus, hepatitis C virus, mycoplasma, bacteria, yeast, and fungi. The culture medium was replaced every 48 hours, and cells at passages 1-3 were used for experiments. For passaging, cells were rinsed once with PBS and incubated with 0.25% trypsin at 37 °C for 1-3 minutes until rounding was observed; digestion was then quenched with complete medium and cells were reseeded. MGSMCs were subjected to high-glucose settings for 72 hours as part of high-glucose modeling, with a normal-glucose group serving as a control. Cells were exposed to TWAD-containing serum in high-glucose conditions for 48 hours as part of the pharmacological intervention, after which samples were taken for further analysis.

Cell viability assessment

MGSMCs were seeded at a density of 1.6 × 10⁴ cells/well into 96-well plates. Following cell attachment, cultures were exposed to high-glucose environments for the specified amount of time, either with or without TWAD therapy. Each well was then filled with 10 μL of CCK-8 solution (G1613-5ML, Servicebio, Wuhan, China) and incubated at 37 °C in 5% CO₂ for one to two hours while shielded from light. A microplate reader (Synergy H1, BioTek, United States) was used to measure absorbance at 450 nm.

Flow cytometric apoptosis assay

Apoptosis was assessed by flow cytometry using Annexin V-FITC/propidium iodide staining. MGSMCs were harvested (including floating cells), washed with PBS, and resuspended in binding buffer. Cells were stained with Annexin V-FITC and propidium iodide at room temperature in the dark according to the manufacturer’s instructions, then immediately analyzed on a flow cytometer. Flow cytometric data were processed using FlowJo software.

Western blot

Total protein was isolated from gastric tissues and GSMCs with RIPA lysis buffer (P0013C, Beyotime, China), and concentrations were determined using a BCA assay kit (ZJ102, Yeasen Biotech, China). Proteins were separated electrophoretically at 80 V through the stacking gel and 120 V in the resolving gel, then transferred to polyvinylidene fluoride membranes. After blocking with 5% non-fat milk in TBST for 1 hour, membranes were incubated overnight at 4 °C with primary antibodies against: Cox6a2 (PA5-104291, Thermo, United States; 11421-1-AP, Proteintech, China), MFN2 (12186-1-AP, Proteintech, China), DRP1 (12957-1-AP, Proteintech, China), SCF (26582-1-AP, Proteintech, China), c-Kit (18696-1-AP, Proteintech, China), and β-actin (66009-1-Ig, Proteintech, China). Following TBST washes, membranes were probed with secondary antibodies for 2 hours at room temperature. Protein bands were visualized with an ECL chemiluminescence kit (Thermo Scientific, United States) and quantified using ImageJ software, with β-actin serving as the loading control.

Immunofluorescence

MGSMCs grown on poly-L-lysine–coated coverslips were fixed with 4% paraformaldehyde for 15 minutes at room temperature and permeabilized with 0.3% Triton X-100 for 10 minutes. After blocking with 5% bovine serum albumin for 1 hour, cells were incubated with rabbit anti-COX6A2 antibody (11421-1-AP, Proteintech, China) overnight at 4 °C, followed by an Alexa Fluor-conjugated goat anti-rabbit secondary antibody for 1 hour at room temperature in the dark. Nuclei were counterstained with DAPI for 10 minutes. Fluorescence images were acquired using a fluorescence microscope with identical acquisition settings across groups.

Measurement of intracellular reactive oxygen species by flow cytometry

Intracellular reactive oxygen species (ROS) was quantified by flow cytometry using a ROS detection kit (E004-1-1, Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions. MGSMCs were detached, collected, and washed with pre-cooled PBS. Cells were incubated with DCFH-DA (D6470, Solarbio, China) working solution prepared in serum-free medium at 37 °C for 20 minutes in the dark, with gentle mixing every 3-5 minutes. After incubation, cells were washed three times to remove excess probe, resuspended in PBS, and immediately analyzed by flow cytometry.

ATP detection

Intracellular ATP levels were measured using an ATP Assay Kit (A095-1-1, Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s specifications. Following the specified treatments, MGSMCs were lysed using the ATP extraction solution included in the kit and rinsed with ice-cold PBS. The ATPase activity was then stopped by heat inactivation (boiling water bath, 10 minutes). After cooling on ice, samples were centrifuged at 12000 × g for 5-10 minutes at 4 °C. The supernatants were then collected to measure ATP. A standard curve was used to calculate the ATP content, which was then normalized to the total protein concentration and reported as μmol/gprot.

Mitochondrial transmembrane potential measurement

Mitochondrial membrane potential (ΔΨm) was measured using the JC-1 Mitochondrial Membrane Potential Assay Kit (C2003S, Beyotime, China) according to the manufacturer’s instructions. MGSMCs were seeded in 6-well plates and treated as indicated, then incubated with JC-1 working solution (1:200) at 37 °C for 20 minutes in the dark. After washing with the kit buffer, cells were immediately examined by fluorescence microscopy. ΔΨm was expressed as the red/green fluorescence ratio (JC-1 aggregates/monomers).

RESULTS

Chemical components of TWAD and its serum-absorbed constituents

UHPLC-Q-Orbitrap HRMS was used to examine the chemical components of TWA. Figure 1A and B, and Supplementary Table 1 display the base peak ion chromatograms and TWA identification results, respectively. A total of 93 components of the TWA decoction were identified. Furthermore, Figure 1C and D, and Supplementary Table 2 display the base peak ion chromatograms and identification results for the components that were absorbed into the serum. A total of 32 compounds were detected in TWAD-containing serum, comprising 9 prototype constituents and 23 metabolites. The prototype constituents comprised including Nobiletin, atractylenolide II, Guvacine, 5-o-feruloylquinic acid, Vicenin 2, Rhein-8-o-β-d-glucopyranoside, Naringenin, Apigenin and Isosinensetin.

Figure 1 The ultra-performance liquid chromatography coupled with a hybrid quadrupole-Orbitrap high-resolution mass spectrometer analysis results of Tangwei’an. A and B: Base peak ion chromatograms of Tangwei’an were acquired in positive and negative ion modes; C and D: Corresponding chromatograms from the Tangwei’an-containing serum analysis were obtained under identical conditions.

TWA alleviates pathological injury and improves gastrointestinal motility in DGP mice

Compared with db/m mice, db/db mice showed significantly reduced GE rate (42% vs 81%, P < 0.01) and small intestinal propulsion rate (40% vs 66%, P < 0.01), indicating successful establishment of the DGP model (n = 5 per group, Supplementary Figure 1). To assess the therapeutic potential of TWA against DGP, graded doses were administered daily following successful DGP model establishment (Figure 2A). During the study’s first phase, every animal stayed healthy. Mice in the Model group experienced typical symptoms such as rough hair, fatigue, decreased mobility, abdominal distension, and changed stool consistency once the model was successfully established. Mortality throughout the treatment phase resulted in final group sizes of: Control (n = 8), Model (n = 8), TWA-H (n = 9), TWA-M (n = 8), TWA-L (n = 7), and Mosapride (n = 9), with recorded deaths of 2, 2, 1, 1, and 3 animals in the Model, TWA-M, TWA-H, Mosapride, and TWA-L groups, respectively. Prior to intervention, all experimental groups exhibited significantly higher body weight and fasting blood glucose levels compared to the Control group (P < 0.01), with no significant differences among these groups. Throughout the 8-week treatment period, body weight increased progressively in all groups, while fasting blood glucose remained persistently elevated in experimental groups relative to Controls (P < 0.01 at all timepoints). Neither TWA nor mosapride treatment significantly altered body weight or glucose levels compared to the Model group (Figure 2B-D).

Figure 2 Tangwei’an alleviates pathological injury and improves gastrointestinal motility in diabetic gastroparesis mice. A: Experimental workflow of the diabetic gastroparesis model and treatment procedure; B: Fasting blood glucose levels of mice at different time points (n = 7-9); C: Body weight of mice at different time points (n = 7-9); D: Representative photographs of mice from each experimental group; E: Gastric emptying rate of each group (n = 7-9); F: Intestinal transit rate of each group (n = 7-9); G: Hematoxylin & eosin staining of gastric tissues in each group. Scale bar = 100 μm; H: Gastric histopathological score (n = 3); I: Hematoxylin & eosin staining of colonic tissues in each group. Scale bar = 100 μm; J: Colonic histopathological score (n = 3); K: Serum levels of Motilin, serotonin, substance P, cholecystokinin, and vasoactive intestinal peptide in mice (n = 7-9). Data are presented as the means ± SEM. ^aP < 0.05 vs Control group, ^bP < 0.01 vs Control group, ^cP < 0.05 vs the Model group, ^dP < 0.01 vs the Model group. TWA-H: Tangwei’an decoction high-dose group; TWA-M: Tangwei’an decoction medium-dose group; TWA-L: Tangwei’an decoction low-dose group; 5-HT: Serotonin; SP: Substance P; CCK: Cholecystokinin; VIP: Vasoactive intestinal peptide.

GE and small intestinal propulsion rates were significantly reduced in the Model group (P < 0.01), whereas all treatment groups showed significant improvement. TWA-H and TWA-M produced the most pronounced enhancement, indicating a clear dose-dependent effect of TWA in restoring gastrointestinal motility (Figure 2E and F). Histological examination and semi-quantitative scoring revealed progressive structural improvements in both gastric and colonic tissues following TWA treatment. Gastric sections from the Model group showed disrupted glandular architecture with inflammatory infiltration and a significantly increased gastric histopathological score, whereas the TWA-H group exhibited near-normal gastric pit arrangement, markedly reduced inflammation, and a lower gastric histopathological score. Similarly, colonic tissues from the TWA-H and TWA-M groups maintained relatively intact crypt structures with abundant goblet cells and showed lower colonic histopathological scores than the Model group, which displayed evident mucosal damage and inflammatory response. The Mosapride group showed partial histological improvement despite persistent submucosal edema (Figure 2G-J).

The Model group had lower levels of motilin, 5-HT, and SP compared to the Control group (P < 0.05), but higher levels of CCK and VIP (P < 0.01). TWA-H and TWA-M dramatically boosted motilin, while mosapride raised 5-HT. TWA-H restored SP levels. Furthermore, CCK levels decreased in the TWA-H, TWA-M, and Mosapride groups, although VIP was repressed across all treatments. These findings indicate that TWA, particularly at high dosages, can effectively correct dysregulated gastrointestinal hormones in diabetic mice (Figure 2K).

Proteomics analysis of TWA intervention in DGP

Next, proteomics sequencing was applied to further explore the specific mechanisms by which TWA ameliorated DGP. DEPs in the gastric tissues in 3 groups (Control, Model, and TWA 21.32 g/kg) were detected. PCA of DEPs revealed distinct clustering patterns among experimental groups. Individual samples are represented as points colored by group (Control: Orange; Model: Green; TWA: Blue), with clear separations observed between Model/Control and TWA/Model groups, demonstrating significant proteomic differences (Figure 3A and B).

Figure 3 Proteomics analysis of Tangwei’an treated diabetic gastroparesis mice. A: Principal component analysis score plot of Model and Control groups; B: Principal component analysis score plot of Tangwei’an (TWA) and Model groups; C: Venn diagram of differentially expressed proteins (DEPs); D: Volcano plots illustrating DEPs of Model vs Control; E: Volcano plots illustrating DEPs of TWA vs Control; F: The UpSet intersection diagram presented the common protein distribution of four types; G: Gene Ontology enrichment analysis of differentially expressed proteins between the TWA and Model groups, including biological process, cellular component, and molecular function; H: Gene Ontology analysis diagram of diabetic gastroparesis for TWA treatment of diabetic gastroparesis; I: Kyoto Encyclopedia of Genes and Genomes enrichment analysis among TWA and Model; J: Kyoto Encyclopedia of Genes and Genomes enrichment chord diagram. TWA: Tangwei’an; KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene Ontology; PCA: Principal component analysis.

Proteomics sequencing was then used to investigate the precise mechanisms through which TWA improved DGP. Three groups (Control, Model, and TWA-H) had DEPs found in their gastric tissues. A total of 1244 (322 upregulated and 922 downregulated proteins in Model) and 3690 (2296 upregulated and 1590 downregulated proteins in TWA) DEPs were found after screening between the Model and Control groups and between the Model and TWA groups based on the q < 0.05 and |log2FC| > 1 criteria. Venn analysis showed that 225 initially elevated proteins were considerably suppressed, while 780 proteins that were downregulated in the Model group were significantly increased following TWA treatment (Figure 3C-F). Potentially important targets through which TWA reduces DGP are these reversibly expressed proteins.

GO and KEGG enrichment analysis were performed on these 1005 DEPs to find functional pathways that were significantly enriched. According to the results of the GO analysis, the biological processes that TWA controlled in DGP primarily comprised proton motive force-driven mitochondrial ATP synthesis, mitochondrial respiratory chain complex IV assembly, and mitochondrial electron transport, cytochrome C to oxygen. The cellular components were enriched in cytosol, lysosome, and mitochondrion. Key molecular functions terms included RNA binding, actin binding, and proton-transporting ATP synthase complex (Figure 3G). A circular diagram of the top 20 enriched GO keywords by P value is shown in Figure 3H. Based on KEGG pathway analysis, TWA may ameliorate DGP primarily through the OXPHOS pathway (Figure 3I). Key DEPs identified in this pathway include Mtnd1, Mtnd5, Mtco1, and Cox6a2, as visualized in the KEGG enrichment chord diagram (Figure 3J).

Metabonomics analysis of TWA intervention in DGP

Metabolomic analysis of gastric tissues from DGP model mice was conducted using multivariate statistical approaches. PCA demonstrated clear metabolic separation between the Control and Model groups (Figure 4A), indicating substantial metabolic disturbances. TWA intervention significantly altered the metabolic profiles, forming a distinct cluster separate from both Control and Model groups and demonstrating its regulatory effects. To further enhance group discrimination, supervised PLS-DA was employed (Figure 4B). This analysis significantly improved separation among the three groups, with each group forming compact clusters in distinct regions of the score plot. The model exhibited high explanatory and predictive power, with R2Y and Q2 values of 0.992 and 0.894 for Model vs Control, and 0.995 and 0.902 for TWA vs Model, respectively. These near-unity values indicate excellent model predictability. Furthermore, OPLS-DA confirmed the metabolic separation among groups (Figure 4C), with all data points located within the 95% confidence interval. Model reliability was rigorously validated through 200 permutation tests, which demonstrated clear separation between the original and permuted models. The R2 values [(0, 0.889) for Model vs Control; (0, 0.856) for TWA vs Model] consistently exceeded the corresponding Q2 values [(0, -0.71) and (0, -0.598)]. The negative Q2 intercepts confirmed the absence of overfitting, thereby establishing the statistical reliability of all models.

Figure 4 Metabonomics analysis of Tangwei’an treated diabetic gastroparesis mice. A: Principal component analysis of metabolites; B: PLS-DA analysis of metabolites; C: OPLS-DA analysis model of intergroup differential metabolites; D: Volcano plot of differentially expressed metabolites in metabonomics; E: Heatmap visualization of metabolites in Control, Model, and Tangwei’an groups; F: Venn and UpSet diagram analysis of differentially expressed metabolites; G: Lollipop plot of metabolites in Tangwei’an decoction-treated diabetic gastroparesis; H: Kyoto Encyclopedia of Genes and Genomes analysis of differentially expressed metabolites in Tangwei’an decoction-treated diabetic gastroparesis. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. TWA: Tangwei’an; VIP: Vasoactive intestinal peptide.

A comparative examination of the Model and Control groups revealed 200 DEMs, including 108 upregulated and 92 downregulated metabolites. In contrast, 432 DEMs were found in the TWA vs Model comparison, with 216 metabolites upregulated and an equivalent amount downregulated. These difference metabolites were visualized using volcano plots and heatmaps (Figure 4D and E). Venn diagrams were utilized to find common DEMs among groups based on the DEMs study. The results showed that 34 metabolites down-regulated in the Model group were significantly up-regulated after TWA treatment, including PE(P-18:0/0:0), fumaric acid, phosphoric acid, D-glucuronic acid 1-phosphate, and 9-tetradecenoic acid. Conversely, 50 metabolites up-regulated in the Model group were significantly down-regulated in the TWA group, such as ginkgolide A, 2,5-dioxopentanoate, gamma-delta-dioxovaleric acid, PE[14:0/14:1(9Z)], and 3-methyl pyruvic acid. The top 10 metabolites with the highest VIP scores were visualized using lollipop plots (Figure 4F and G). KEGG enrichment analysis revealed that the DEMs were primarily involved in OXPHOS, lysosome, apoptosis, and the FoxO signaling pathway (Figure 4H).

Integrated proteomics-metabolomics analysis identifies COX6A2 as a key node in TWAD-regulated OXPHOS

To integrate the proteomic and metabolomic alterations induced by TWAD, we performed a joint pathway analysis using DEPs and DEMs. Intersection analysis via Venn diagrams revealed seven common KEGG pathways (Figure 5A). Among these, OXPHOS emerged as the most prominently altered pathway across both molecular levels, as visualized in the KEGG bubble plot (Figure 5B), consistent with the pivotal role of OXPHOS in the pathogenesis and progression of diabetes and its complications[24]. We further performed correlation analysis between significantly altered proteins and metabolites in DGP mice following TWA treatment. The results revealed that proteins P00397 (Mtco1), P03888 (Mtnd1), P03921 (Mtnd5), and P43023 (Cox6a2) all showed significant negative correlations with metabolite ADP, while demonstrating significant positive correlations with metabolites fumaric acid and phosphoric acid. COX6A2 was the major node in the integrated network and showed the highest metabolic connections among these proteins (Figure 5C and D). To further clarify the mechanistic relevance of COX6A2 within the integrated network, we conducted molecular docking with the major active components of TWAD. Nobiletin and atractylenolide II showed favorable binding with COX6A2 (Figure 5E and F), and the detailed docking results are summarized in Supplementary Table 3. COX6A2 is a key subunit of cytochrome C oxidase that drives OXPHOS and is essential for maintaining normal mitochondrial structure and function[25]. These multi-omics and docking analyses support COX6A2 as a prioritized candidate target underlying TWAD-driven improvement of mitochondrial OXPHOS, thereby linking the multi-omics signature to downstream mitochondrial dynamics and setting the stage for mechanistic validation in vivo and in vitro.

Figure 5 Integrated analysis of proteomics and metabolomics. A: Venn diagram illustrating the overlapping Kyoto Encyclopedia of Genes and Genomes pathways enriched by differentially expressed proteins (DEPs) and differentially expressed metabolites (DEMs); B: Integrated proteomic and metabolomic Kyoto Encyclopedia of Genes and Genomes bubble plot; C: Analyzing the correlation between DEPs and DEMs; D: Correlation network map of DEPs and DEMs; E: Molecular docking of COX6A2 with Nobiletin; F: Molecular docking of COX6A2 with atractylenolide II. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001.

TWAD regulates mitochondrial dynamics in DGP mice via modulation of COX6A2

Mitochondrial dysfunction is closely associated with delayed GE in diabetic gastroenteropathy[10]. SCF/c-Kit signaling drives gastrointestinal motility signal transduction, and its dysregulation contributes to peristaltic dysfunction[12]. To determine the effect of TWAD treatment on mitochondrial homeostasis in DGP mice, we looked at COX6A2 expression together with SCF and c-Kit levels, gastric ATP level, and the mitochondrial dynamics markers DRP1 and MFN2. Western blot analysis revealed that COX6A2, SCF, and c-Kit protein levels were significantly decreased in the Model group compared with the Control group (P < 0.01). Both TWA and mosapride significantly restored COX6A2, SCF, and c-Kit expression relative to the Model group (P < 0.01, Figure 6A-D). Regarding mitochondrial dynamics, the Model group exhibited increased DRP1 and decreased MFN2 compared with the Control group (P < 0.01). TWA and mosapride each significantly reduced DRP1 and increased MFN2 relative to the Model group (P < 0.01, Figure 6E-G). Consistently, gastric ATP content was significantly reduced in the Model group whereas ATP levels were significantly elevated in both the TWA and Mosapride groups compared with the Model group (P < 0.01, Figure 6H).

Figure 6 Tangwei’an regulates mitochondrial dynamics in diabetic gastroparesis mice via modulation of COX6A2. A-D: Representative western blots and densitometric quantification of COX6A2, SCF and c-Kit in gastric tissues (n = 3); E-G: Representative western blots and densitometric quantification of DRP1 and MFN2 in gastric tissues (n = 3); H: ATP content in mice gastric tissues (n = 6). Data are presented as the mean ± SEM. ^bP < 0.01 vs Control group, ^dP < 0.01 vs the Model group. TWA: Tangwei’an.

COX6A2/SCF/c-Kit signaling mediates TWAD-regulated mitochondrial dynamics in high glucose-injured MGSMCs

To validate the in vivo findings and define the molecular relationship between COX6A2, SCF/c-Kit signaling, and mitochondrial dynamics, we developed a high glucose (HG)-injured MGSMC model and treated cells with TWAD-containing serum. A 35 mmol/L glucose condition was selected as the in vitro modeling concentration based on glucose-dose screening using the CCK-8 assay. TWAD-containing serum was subsequently titrated, and 6% was determined as the optimal dose using CCK-8 and flow cytometric apoptosis analysis (Supplementary Figure 2), with 6% selected for subsequent experiments. HG substantially decreased ATP synthesis (Figure 7A) and increased intracellular ROS levels (Figure 7B and C) compared with the Control group. JC-1 staining indicated mitochondrial depolarization, with increased monomers (green), decreased aggregates (red), and a reduced red/green ratio (Figure 7D). DRP1 was upregulated and MFN2 was downregulated under HG conditions (Figure 7E). TWAD-containing serum markedly reversed these changes, restoring ATP levels, lowering ROS, improving ΔΨm, and normalizing DRP1/MFN2 expression toward the control profile (Figure 7).

Figure 7 Tangwei’an rescues mitochondrial dysfunction in high glucose-injured mouse gastric smooth muscle cells. A: ATP content in mouse gastric smooth muscle cells (n = 6); B: Reactive oxygen species; C: Reactive oxygen species levels quantified as MFI (a.u.); D: Representative JC-1 images and quantification of mitochondrial membrane potential as the red/green ratio (a.u.); E: Representative western blots and densitometric quantification of DRP1 and MFN2 in mouse gastric smooth muscle cells (n = 3). Data are presented as the mean ± SEM. ^bP < 0.01 vs Control group, ^dP < 0.01 vs the Model group. TWA: Tangwei’an; ROS: Reactive oxygen species.

We next examined whether COX6A2 mediates TWAD effects on SCF/c-Kit signaling and mitochondrial dynamics. Immunofluorescence showed that TWAD-containing serum increased COX6A2 signal under HG, and this increase was blunted by COX6A2 knockdown, whereas COX6A2 overexpression enhanced COX6A2 signal (Figure 8A). Western blot quantification confirmed the corresponding changes in COX6A2 protein levels (Figure 8B and C). SCF and c-Kit expression were lowered by HG, and their levels were restored by TWAD-containing serum; COX6A2 knockdown reduced this restoration, whereas COX6A2 overexpression partially replicated the TWAD effect by raising SCF and c-Kit under HG conditions (Figure 8B, D and E). TWAD-containing serum reduced DRP1 and increased MFN2 in HG-injured MGSMCs, and these changes were blunted by COX6A2 knockdown; COX6A2 overexpression shifted DRP1/MFN2 toward the TWAD-like pattern (Figure 8B, F and G). ATP generation was enhanced by TWAD-containing serum, but this improvement was diminished by COX6A2 knockdown (Figure 8H). These findings support a COX6A2-dependent pathway that links TWAD to SCF/c-Kit signaling and mitochondrial dynamics in HG-injured MGSMCs.

Figure 8 COX6A2 links Tangwei’an to SCF/c-Kit signaling and mitochondrial dynamics in mouse gastric smooth muscle cells. A: Representative immunofluorescence images of COX6A2 in the indicated groups; B-G: Representative western blots and densitometric quantification of COX6A2, SCF, c-Kit, DRP1 and MFN2 in mouse gastric smooth muscle cells (n = 3); H: ATP content in mouse gastric smooth muscle cells (n = 6). ^eP < 0.01 vs the “high glucose(-) Tangwei’an(-)” group, ^fP < 0.05 vs the “high glucose(+) Tangwei’an(-)” group, ^gP < 0.01 vs the “high glucose(+) Tangwei’an(-)” group. HG: High glucose; TWA: Tangwei’an.

DISCUSSION

DGP has grown to be a significant challenge to public health and healthcare funding, significantly lowering patients’ quality of life while also seriously harming their mental health[26]. Clinical evidence suggests that TCM interventions can effectively alleviate key symptoms of DGP and improve gastric motility, with favorable safety and tolerability[9,27]. TWAD was developed by Professor Xiao-Lin Tong and colleagues on the basis of long-term clinical practice in diabetes and its complications, and was formulated through modification of classical prescriptions including Xiaobanxia Decoction, Zhizhu Decoction, and Dahuang Huanglian Xiexin Decoction. It consists of eight herbs: Zhishi (Citrus aurantium L.), Baizhu (Atractylodes macrocephala Koidz.), Binlang (Areca catechu L.), Dahuang (Rheum officinale Baill.), Banxia [Pinellia ternata (Thunb.) Breit.], Shengjiang (Zingiber officinale Rosc.), Taoren [Prunus persica (L.) Batsch], and Dangshen [Codonopsis pilosula (Franch.) Nannf.][28]. The formulation reflects the traditional rationale of targeting spleen-stomach dysfunction by restoring disordered qi movement and the ascending-descending balance that underlies nausea, vomiting, and postprandial fullness in DGP. To clarify the bioactive material basis, TWAD medicated serum was prepared and profiled by UHPLC-Q-Orbitrap HRMS[29], identifying 9 prototype constituents and 23 metabolites. Prior studies have shown that atractylenolide improves abnormal GE and gastric motility in diabetic gastrointestinal dysfunction models, with reported involvement of SCF/c-Kit signaling and oxidative stress regulation[12]. Nobiletin, naringenin, and isosinensetin, representative polymethoxyflavonoids from Citrus aurantium, display anti-inflammatory and glycometabolic regulatory activities[30,31], and nobiletin has been reported to support glycaemic homeostasis and β-cell protection through modulation of gut microbiota and metabolic homeostasis[32]. 5-O-feruloylquinic acid has been reported to ameliorate glucose metabolic disturbances in insulin-resistant HepG2 cells via the phosphoinositide 3-kinases/protein kinase B pathway[33]. These findings point to the bioactive material basis of TWAD and suggest that its therapeutic effects may arise from coordinated actions of multiple constituents.

To evaluate the therapeutic efficacy of TWAD, a DGP model was established in db/db mice based on our previous work[20]. Db/db mice are widely used in studies of diabetic gastrointestinal dysfunction because they spontaneously develop obesity, persistent hyperglycemia, and metabolic disturbances that recapitulate the background of type 2 diabetes. Previous studies have shown that C57BLKS/J db/db mice exhibit altered GE and regional gastric contractility and have therefore been used as an experimental model for DGP and related gastric motility disorders[34]. Mosapride was used as a positive control to benchmark prokinetic efficacy and was selected as a 5-HT4 receptor agonist that promotes gastrointestinal motility by enhancing acetylcholine release from excitatory neurons in the stomach and duodenum[35,36]. Gastric and colonic histology in the db/db DGP model showed marked mucosal injury with inflammatory infiltration, which was accompanied by delayed GE and reduced intestinal transit. Both TWAD and mosapride attenuated these pathological and functional abnormalities, and high-dose TWAD produced a more pronounced restoration of gastric and colonic mucosal architecture. Neurohumoral imbalance is also implicated in delayed GE. Motilin, 5-HT, and SP generally favour propulsion, whereas CCK and VIP act as inhibitory signals that restrain GE[37-41]. In the db/db DGP model, motilin, 5-HT, and SP were reduced and CCK and VIP were increased. TWAD largely normalised this hormone profile by elevating motilin, 5-HT, and SP while lowering CCK and VIP. These endocrine shifts, together with improved mucosal integrity, are consistent with the recovery of GE and intestinal transit. All things considered these results demonstrate that TWAD improves gastrointestinal function in DGP mice.

Metabolomics has been widely applied to diabetes and its complications research[42-44]. Gastric tissue metabolomics showed that TWAD restored 84 DGP-associated DEMs toward physiological levels in DGP mice, and KEGG pathway annotation mapped these restored metabolites mainly to OXPHOS, the lysosome, and apoptosis-related pathways. Notably, ADP, phosphoric acid, and fumaric acid were identified as key metabolic targets of TWAD, with decreased ADP and increased phosphoric acid and fumaric acid following treatment. ADP is mainly involved in OXPHOS, lysosome, and FoxO signaling pathways and serves as a key metabolite in cellular energy metabolism. During cellular respiration, ADP is phosphorylated to generate ATP, thereby providing essential energy for mitochondrial function[45]. ADP is an endogenous agonist at the P2Y₁ receptor and can mobilize intracellular Ca²⁺ following receptor activation[46]. In the enteric nervous system, ADP has been reported to evoke Ca²⁺ transients in enteric glial cells, and enteric glial Ca²⁺ activity is increasingly recognized as a component of local reflex circuits governing gastrointestinal motility and fluid transport[47,48]. Under oxidative stress, ADP-associated AMP-activated protein kinase activation contributes to energy buffering by modulating FoxO-dependent regulation of arginine kinase, thereby supporting cellular energy homeostasis[49]. Phosphoric acid and fumaric acid also participate in OXPHOS-related processes and contribute to ATP synthesis and turnover, providing essential energetic support[50]. Consistent with our findings, Xiangsha Liujun combined with Banxia Xiexin Decoction has been reported to improve gastric motility in DGP rats by restoring glucose-1-phosphate levels[51].

Proteomic analysis of gastric tissue was conducted to identify TWAD-responsive proteins and to explore potential biomarkers related to DGP. TWAD treatment reversed dysregulated protein expression in DGP mice. GO enrichment analysis of the DEPs indicated predominant associations with proton motive force-driven mitochondrial ATP synthesis, mitochondrial respiratory chain complex IV, and mitochondrial electron transport from cytochrome c to oxygen. Integrative pathway enrichment of DEPs and DEGs further suggested that improvement of OXPHOS may be involved in the effects of TWAD on gastrointestinal symptoms in DGP mice. OXPHOS plays a critical role in the development and progression of diabetes and its complications[24]. Insulin resistance and hyperglycemia can directly impair mitochondrial function, reduce OXPHOS efficiency, and consequently decrease ATP production[52]. Under hyperglycemic conditions, impaired OXPHOS and disordered energy metabolism drive cells to increase glycolysis to compensate for energy demand. This metabolic shift can in turn induce oxidative stress and inflammatory responses[53]. OXPHOS regulates cellular bioenergetics through the coordinated actions of the electron transport chain, ATP synthase, metabolite availability, and signaling pathways. The electron transport chain constitutes the core of OXPHOS by transferring electrons and pumping protons to generate a transmembrane proton gradient that drives ATP synthesis by ATP synthase[54]. Metabolites such as citrate and long-chain fatty acids can modulate electron transport chain activity and ATP synthase function, thereby influencing OXPHOS efficiency[55,56]. Previous studies have shown that AMP-activated protein kinase, mechanistic target of rapamycin, and peroxisome proliferator-activated receptor gamma coactivator-1α signaling pathways regulate mitochondrial apoptosis and energy metabolism in GSMCs in DGP models and are associated with OXPHOS regulation[57-59].

Respiratory chain complex IV (cytochrome C oxidase) is the terminal enzyme complex of the mitochondrial electron transport chain. It catalyzes the transfer of electrons from cytochrome C to oxygen while simultaneously driving proton pumping from the mitochondrial matrix into the intermembrane space[60]. COX6A2 is a key subunit of cytochrome C oxidase that supports OXPHOS and plays an important role in maintaining normal mitochondrial structure and function. Reduced COX6A2 expression leads to abnormal energy metabolism, elevated oxidative stress, altered Ca²⁺ transport activity, and decreased contractile capacity[25,61]. COX6A2 is selectively and highly expressed in PV⁺ interneurons, where it helps maintain energy homeostasis by regulating complex IV activity and dynamically sensing ATP/ADP ratio changes to support high-frequency firing[62]. COX6A2 deficiency has been linked to metabolic disturbance and oxidative stress and to impaired maturation of PV⁺ interneurons[63]. In the present study, COX6A2 expression was significantly reduced in gastric tissue from DGP model mice and was restored toward physiological levels after TWAD treatment. These results indicate that TWAD improves energy metabolism in DGP mice by regulating electron transfer during OXPHOS.

ICCs are the core pacemaker cells responsible for the regulation of rhythmic gastrointestinal motility. Their spindle-shaped cell bodies exhibit distinctive ultrastructural features, including abundant mitochondria and endoplasmic reticulum, expression of multiple membrane channel proteins, and extensive network connections formed through dendritic processes[64,65]. Through the generation of slow-wave electrical activity, ICCs regulate the rhythmicity of smooth muscle contraction, and ICC abundance, network integrity, and mitochondrial functional status are key determinants of coordinated gastrointestinal motility[66,67]. The transmembrane receptor tyrosine kinase c-Kit is selectively expressed in ICCs and plays a critical role in gastrointestinal motility regulation. Activation of c-Kit by its ligand SCF supports ICC maintenance and has been associated with ICC proliferation and structural repair, with consequent effects on gastrointestinal contractility[12]. Previous studies have shown that downregulation of SCF/c-Kit expression in gastric tissue from DGP models is closely associated with ultrastructural injury in ICCs, including mitochondrial cristae disruption and endoplasmic reticulum dilation[68]. Pharmacological inhibition of c-Kit attenuates drug-induced improvements in gastrointestinal dysmotility, indicating that SCF/c-Kit signaling is essential for ICC function[69]. In the present study, COX6A2, SCF, and c-Kit protein levels were reduced in gastric tissue from DGP model mice. TWAD restored all three proteins in a dose-dependent manner, and the between-group differences were significant. These findings align with our omics analyses and suggest improved OXPHOS. Gastric ATP levels were reduced in DGP mice with increased DRP1 and decreased MFN2, and TWAD increased ATP and shifted DRP1/MFN2 toward the control pattern. In MGSMCs, HG reduced ATP and ΔΨm and increased ROS with the same DRP1/MFN2 shift. TWAD-containing serum reversed these changes. It should be noted that no dedicated osmotic control was included in the high-glucose cell model, and thus the possible contribution of osmotic pressure to the observed in vitro changes cannot be completely excluded. COX6A2 knockdown weakened the TWAD effect on SCF and c-Kit and reduced recovery of DRP1/MFN2 and ATP, while COX6A2 overexpression increased SCF and c-Kit and shifted DRP1/MFN2 toward a TWAD-like profile. In vivo and in vitro experiments support that TWAD ameliorates DGP by regulating mitochondrial dynamics through the COX6A2/SCF/c-Kit pathway.

CONCLUSION

This study demonstrates that TWAD ameliorates DGP by regulating mitochondrial dynamics through the COX6A2/SCF/c-Kit signaling pathway (Figure 9). TWAD restores OXPHOS, rebalances DRP1/MFN2, and improves gastrointestinal motility in vivo and in vitro. These findings provide mechanistic support for TWAD as a therapeutic strategy for DGP.

Figure 9 Molecular mechanism of Tangwei’an decoction on improving diabetic gastroparesis. OXPHOS: Oxidative phosphorylation; TWA: Tangwei’an; DGP: Diabetic gastroparesis.